This invention relates to fungal/bacterial antagonist combinations, a seed coated with said combinations and a plant protected from plant pathogens by said combinations. In particular, the invention relates to fungal/bacterial antagonist combinations and their use for controlling plant pathogens.
Early and late season stalk and root rot are major causes of crop loss. A variety of plants are affected, including tomatoes, peppers, turf grass, soybeans, sunflower, wheat and corn, The pathogens that cause these symptoms include fungi of the genera Fusarium, Phythium, Phytophthora and Penicillium.
One approach to solving the problem of early season damping off of plants is treatment of seeds with fungicides, such as captan, metalaxyl and Maxim. Although these chemicals enhance seed germination and seedling stand by inhibiting the pathogenic ability of Phythium spp. (active in cool, wet soils), they have no activity against the pathogenic fungi that are responsible for late season root and stalk rot.
Fusarium and Penicillium are the pathogens responsible for late season root and stalk rot. These pathogens prefer the warm, dry conditions that occur late in the growing season. There is no chemical or biological fungicide available that addresses the problem of late season root and stalk rot in corn. Currently, the only way to deal with this problem is to periodically rotate to a non-susceptible crop to reduce pathogen numbers. Corn growers can also select hybrids that have better xe2x80x9cstandability,xe2x80x9d but such hybrids usually have lower yields. Unfortunately, the corn varieties with the highest yields are usually those most susceptible to late season root and stalk rot.
Trichoderma is a genus of fungi that contains about 20 species. Synonyms for the genus name include Aleurisma and Sporoderma. Trichoderma virens, which is also called Gliocladium virens, is a member of the genus. The natural habitats of these fungi include soil and plant material. A member of the genus, Trichoderma harzianum KRL-AG2 (ATCC 20847) also known as strain T-22, is used as a biocontrol agent that is applied as a seed or soil treatment or on cuttings and transplants. Strains of the species, Trichoderma virens, have also been used for control of damping off diseases in plants. For example, Gliocladium virens GL-21 is sold under the tradename SoilGard(copyright) (formerly GlioGard).
Bacillus is a genus of rod-shaped, gram-positive, aerobic or (under some conditions) anaerobic bacteria. Bacillus species are widely found in soil and water and some have been used to control plant diseases, including root rot. Bacillus amyloliquefaciens is a spore-forming member of the genus. Bacillus amyloliquefaciens L. L. Cambell strain F (ATCC 23350) is the type strain for the species. Other known and commercially available Bacillus amyloliquefaciens strains include those having the following ATCC accession numbers: 23842,23843, 23844 and 23845 (Int. J. Sys. Bacteriol. 37:69-71, 1987; J. Bacteriol. 94:1124-1130, 1967).
Bacillus lentimorbus is another spore-forming member of the genus. Bacillus lentimorbus Dutky 1940 (ATCC 14707) is the type strain for the species (Skerman, V. B. D., McGowan, V., and Sneath, P. H. A., Approved lists of bacterial names. Int. J. Syst. Bacteriol. 30:225-420, 1980). Some researchers consider Bacillus lentimorbus to be a variety of Bacillus popilliae. Bacillus lentimorbus and Bacillus popilliae have recently been reclassified as Paenibacillus lentimorbus and Paenibacillus popilliae (Pettersson, B., Rippere, K. E., Yousten, A. A. and Priest, F. G., Transfer of Bacillus lentimorbus and Bacillus popilliae to the genus Paenibacillus with emended descriptions of Paenibacillus lentimorbus comb. nov. and Paenibacillus popilliae comb. nov., Int. J. yst. Bacterial. 49: 531-540, 1999). Bacillus lentimorbus and Bacillus popilliae are the causative agents of milky disease in Japanese beetles and related scarab larvae and xe2x80x9cmilky sporexe2x80x9d powders are sold under the trade names, xe2x80x9cDoom,xe2x80x9d xe2x80x9cMilky Spore,xe2x80x9d xe2x80x9cJapidemicxe2x80x9d xe2x80x9cGrub Killerxe2x80x9d and xe2x80x9cGrub Attack,xe2x80x9d for biocontrol of these insects.
Background art biocontrol products have comprised the bacterium Burkholderia cepacia, which is also known as Pseudomonas cepacia. This bacterium has been implicated as a human pathogen. Furthermore, it has little or no shelf life unless refrigerated at 4 degrees Centigrade at a minimum of 20 percent moisture.
The background art is characterized by U.S. Pat. Nos. 4,476,881; 4,489,161; 4,642,131; 4,668,512; 4,678,669; 4,713,342; 4,724,147; 4,748,021; 4,818,530; 4,828,600; 4,877,738; 4,915,944; 4,952,229; 5,047,239; 5,049,379; 5,071,462; 5,068,105; 5,084,272; 5,194,258; 5,238,690; 5,260,213; 5,266,316; 5,273,749; 5,300,127; 5,344,647; 5,401,655; 5,422,107; 5,455,028; 5,409,509; 5,552,138; 5,589,381; 5,614,188; 5,628,144; 5,632,987; 5,645,831; 5,665,354; 5,667,779; 5,695,982; 5,702,701; 5,753,222; 5,852,054; 5,869,042; 5,882,641; 5,882,915; 5,906,818; 5,916,029; 5,919,447; 5,922,603; 5,972,689; 5,974,734; 5,994,117; 5,998,196; 6,015,553; 6,017,525; 6,030,610; 6,033,659; 6,060,051; and 6,103,228; the disclosures of which patents are incorporated by reference as if fully set forth herein.
Gravely et al. in U.S. Pat. No. 4,476,881 disclose a mixed complementary culture of bacteria and fungi that is used to degrade pectin and cellulose components of tobacco materials. The invention is limited in that it teaches use of different microorganisms for a different purpose.
Papavizas in U.S. Pat. No. 4,489,161 discloses a strain of the fungus Trichoderma viride that is an effective biocontrol agent for fusarium wilt of chrysanthemum. The invention is limited in that it teaches use of a different microorganism.
Hoitinik in U.S. Pat. No. 4,642,131 discloses a process for production of a disease-suppressive compost and a microorganism culture for use therein. The invention is limited in that it teaches use of different microorganisms for a different purpose.
Lewis et al. in U.S. Pat. No. 4,668,512 disclose a method for preparing pellets containing living biocontrol fungi and nutrients. The invention is limited in that it teaches a process that involves use of different microorganisms.
Ricard in U.S. Pat. No. 4,678,669 discloses a method of using immunizing commensals to control soil-borne pathogens. The invention is limited in that it teaches use of different microorganisms.
Chet et al. in U.S. Pat. No. 4,713,342 disclose a novel isolate of Trichoderma and it use. The invention is limited in that it teaches use of a different microorganism.
Marois et al in U.S. Pat. No. 4,724,147 disclose a method for preparing pellets containing living biocontrol fungi. The invention is limited in that it teaches a process that involves use of different microorganisms.
Chet et al. in U.S. Pat. No. 4,748,021 disclose antifungal compositions containing Trichoderma active against Fusarium. The invention is limited in that it teaches use of a different microorganism.
Marois et al. in U.S. Pat. No. 4,818,530 disclose a method for preparing pellets containing living biocontrol fungi. The invention is limited in that it teaches a process that involves use of different microorganisms.
McCabe et al. in U.S. Pat. No. 4,828,600 disclose a biological inoculant for corn. The invention is limited in that it teaches use of different microorganisms.
Handelsman et al. in U.S. Pat. No. 4,877,738 disclose a new microorganism culture and a method for biological control of damping off and root rot. The invention is limited in that it teaches a process that involves use of a different microorganism, Bacillus cereus. 
Chet et al. in U.S. Pat. No. 4,915,944 disclose a novel isolate of Trichoderma and fungicidal compositions containing it. The invention is limited in that it teaches use of a different microorganism.
Muir in U.S. Pat. No. 4,952,229 discloses a soil and foliar supplement. The invention is limited in that it teaches use of different microorganisms.
Pusey in U.S. Pat. No. 5,047,239 discloses a biological control agent for fruit rot. The invention is limited in that it teaches use of a different microorganism for a different purpose.
Handelsman et al. in U.S. Pat. No. 5,049,379 disclose a fungicidal toxin and a method and an inoculum for controlling root rot and damping off. The invention is limited in that it teaches use of a different microorganism, Bacillus cereus. 
Kimura in U.S. Pat. No. 5,071,462 discloses a method and apparatus for producing an organic fertilizer. The invention is limited in that it teaches use of different microorganisms for a different purpose.
Lewis et al. in U.S. Pat. No. 5,069,105 disclose a fungal formulation for biocontrol of soil-borne plant pathogens. The invention is limited in that it teaches use of different microorganisms.
Speakman et al. in U.S. Pat. No. 5,084,272 disclose a Trichoderma fungus and a fungicide that contains it. The invention is limited in that it teaches use of a different microorganism.
Pauu et al. in U.S. Pat. No. 5,194,258 disclose a method for producing enhanced biocontrol agents. The invention is limited in that it teaches use of different microorganisms.
Elad et al. in U.S. Pat. No. 5,238,690 disclose a novel Trichoderma culture and biological compositions containing it. The invention is limited in that it teaches use of different microorganisms for a different purpose.
Harman et al. in U.S. Pat. No. 5,260,213 disclose fused biocontrol agents. The invention is limited in that it teaches use of different microorganisms.
Elad et al. in U.S. Pat. No. 5,266,316 disclose a novel isolate of Trichoderma harzianum and fungicidal compositions containing it. The invention is limited in that it teaches use of different microorganisms for a different purpose.
Bok et al. in U.S. Pat. No. 5,273,749 disclose a process for preparing coated microbial pesticides and the products of the process. The invention is limited in that it teaches use of different microorganisms.
Williams in U.S. Pat. No. 5,300,127 discloses seed coatings. The invention is limited in that it teaches use of different microorganisms.
Rossall in U.S. Pat. No. 5,344,647 discloses a Bacillus strain that has antimicrobial activity. The invention is limited in that it teaches use of different microorganisms.
Kijima et al. in U.S. Pat. No. 5,401,655 disclose a process for biologically preventing plant diseases. The invention is limited in that it teaches use of different microorganisms for a different purpose.
Kubota in U.S. Pat. No. 5,422,107 discloses a novel fungus and a fungicide containing it. The invention is limited in that it teaches use of a different microorganism.
O""Donnell in U.S. Pat. No. 5,455,028 discloses a method of inhibiting fungi. The invention is limited in that it teaches use of a different microorganism.
Burth et al. in U.S. Pat. No. 5,409,509 disclose a seed treatment. The invention is limited in that it teaches use of different microorganisms.
Handelsman et al. in U.S. Pat. No. 5,552,138 disclose a novel strain of Bacillus cerus and a method of protecting plants with the strain. The invention is limited in that it teaches use of a different microorganism.
Neyra et al. in U.S. Pat. No. 5,589,381 disclose a novel strain of Bacillus licheniformis that produces an antifungal agent and a use for the strain. The invention is limited in that it teaches use of a different microorganism.
Urano et al. in U.S. Pat. No. 5,614,188 disclose an anti-Fusarium composition containing strains of Bacillus sp. The invention is limited in that it teaches use of different microorganisms.
Eastin in U.S. Pat. No. 5,628,144 discloses solid matrix priming of seeds. The invention is limited in that it teaches use of different microorganisms.
Payne et al. in U.S. Pat. No. 5,632,987 disclose a Bacillus thuringiensis toxin that is active against corn rootworm larvae. The invention is limited in that it teaches use of a different microorganism for a different purpose.
Chilcott et al. in U.S. Pat. No. 5,645,831 disclose a Bacillus thuringiensis strain and metabolite that are active against corn rootworm. The invention is limited in that it teaches use of a different microorganism for a different purpose.
Neyra et al. in U.S. Pat. No. 5,665,354 disclose a novel strain of Bacillus licheniformis that produces an antifungal agent and a use for the strain. The invention is limited in that it teaches use of a different microorganism.
Kubo in U.S. Pat. No. 5,667,779 discloses a fungi-inhibiting composition comprising Bacillus subtilis. The invention is limited in that it teaches use of a different microorganism.
Handelsman et al. in U.S. Pat. No. 5,695,982 discloses canavanine-resistant strains of Bacillus cereus. The invention is limited in that it teaches use of different microorganisms.
O""Donnell in U.S. Pat. No. 5,702,701 discloses a process for treatment of soil and plants with a composition containing Bacillus laterosporus. The invention is limited in that it teaches use of a different microorganism.
Marrone et al. in U.S. Pat. No. 5,753,222 disclose an antibiotic-producing strain of Bacillus and methods of controlling plant diseases with it. The invention is limited in that it teaches use of a different microorganism.
Handelsman et al. in U.S. Pat. No. 5,852,054 disclose fungicidal toxins from a biocontrol bacterium. The invention is limited in that it teaches use of a different microorganism.
Marrone et al. in U.S. Pat. No. 5,869,042 disclose methods for controlling above-ground plant diseases. The invention is limited in that it teaches use of different microorganisms for a different purpose.
Shetty in U.S. Pat. No. 5,882,641 discloses fruit pomice compositions and their uses. The invention is limited in that it teaches use of different microorganisms.
Howell in U.S. Pat. No. 5,882,915 discloses viridiol-deficient strains of Trichoderma virens and process for making and using biocontrol agents that contain them. The invention is limited in that it teaches use of different microorganisms.
Heins et al. in U.S. Pat. No. 5,906,818 disclose a Bacillus mycoides strain for controlling corn rootworm. The invention is limited in that it teaches use of a different microorganism for a different purpose.
Smith et al. in U.S. Pat. No. 5,916,029 disclose a process for producing seeds coated with a microbial composition. The invention is limited in that it teaches use of different microorganisms.
Marrone et al. in U.S. Pat. No. 5,919,447 disclose a strain of Bacillus for controlling plant disease. The invention is limited in that it teaches use of a different microorganism.
Herrera-Estrella et al. in U.S. Pat. No. 5,922,603 disclose a method for obtaining strains of Trichoderma sp. The invention is limited in that it teaches use of a different microorganism.
Cook et al. in U.S. Pat. No. 5,972,689 disclose methods and compositions for control of root diseases. The invention is limited in that it teaches use of different microorganisms.
Eastin in U.S. Pat. No. 5,974,734 discloses solid-matix priming of seeds with microorganisms. The invention is limited in that it teaches use of different microorganisms.
Handelsman et al. in U.S. Pat. No. 5,998,196 disclose a method for suppressing disease using a novel Bacillus cereus strain. The invention is limited in that it teaches use of a different microorganism.
Germida et al. in U.S. Pat. No. 6,015,553 disclose a Bacillus subtilis strain for controlling pests. The invention is limited in that it teaches use of a different microorganism for a different purpose.
Logan et al. in U.S. Pat. No. 6,017,525 discloses a method of poultry house litter treatment. The invention is limited in that it teaches use of a different microorganism for a different purpose.
Handelsman et al. in U.S. Pat. No. 6,030,610 disclose a novel strain of Bacillus cereus. The invention is limited in that it teaches use of a different microorganism.
Handelsman et al. in U.S. Pat. No. 6,033,659 disclose a novel strain of Bacillus cereus. The invention is limited in that it teaches use of a different microorganism.
Heins et al. in U.S. Pat. No. 6,103,228 discloses compositions and methods for controlling plant pests and a novel strain of Bacillus subtilis. The invention is limited in that it teaches use of a different microorganism.
Branly et al. in U.S. Pat. No. 6,232,270 discloses agricultural compositions containing bacteria. The invention is limited in that it teaches use of different microorganisms and a different process for their use. While the patent suggests that one bacterium that is useful within the disclosed invention is Bacillus amyloliquefaciens, it teaches that spores, cultures or suspensions of the bacterium must be combined with an agriculturally effective ingredient of a plant growth stunting agent or herbicide.
Moesinger in U.S. Pat. No. 6,326,016 discloses plant immunization compositions. The invention is limited in that it teaches use of different microorganisms and a different process for their use. While the patent suggests that one bacterium that can be used according to the disclosed invention is Bacillus amyloliquefaciens, and that one fungi that can be used according to the invention is Trichoderma spp., it teaches production of an extract by means of biotechnological fermentation process performed by only one microorganism. Furthermore, the patent teaches that high-temperature autoclaving (e.g., one hour at +120 degrees C.) and (optionally) filtering are preferred steps in preparation of the extract.
Background art is also disclosed in the non-patent literature. The StratSoy Research Database posted on the Web (apparently in 1996) a description of a research project entitled Interaction of Soybean Roots with Soilborne Pathogens and Nonpathogens. The project studied the use of Bacillus amyloliquefaciens B94 as a biocontrol agent for plant diseases. The invention is limited in that it teaches use of a single different microorganism.
An article entitled Seed""s Coat of Many Microbes Wards Off Rot in Science Update section of the April, 1997, issue of Agricultural Research, disclosed the mixing of helpful fungi and bacteria to weaken or kill fungal pathogens that attack young corn sprouts. The applicant believes that the combination used was T. virens and Psuedomonas cepacia. This invention is limited for two reasons: (1) P. cepacia is implicated as causitive in deaths in cystic fibrosis patients and, therefore, EPA registration would probably not be possible, and (2) P. cepacia is not a spore-forming bacterium and, therefore, the moisture content of the combination must be maintained at greater than about 17 percent. T. virens can be stored and applied at low moisture levels and does not remain in the spore state at the higher moisture levels, making its combination with P. cepacia impractical.
In an article authored by L. E. Datnoff et al. entitled Effect of Bacterial and Fungal Microorganisms to Colonize Tomato Roots, Improve Transplant Growth and Control Fusarium Crown and Root Rot that was posted on the Web in 2000 by the University of Florida Southwest Florida Research and Education Center, the authors discuss using a combination of an unidentifed bacterial strain and Gliocladium virens to control Fusarium root rot. The invention is limited in,that it teaches use of different microorganisms.
The U.S. Environmental Protection Agency Office of Pesticide Programs posted a Biopesticide Fact Sheet on Bacillus subtilis var. amyloliquefaciens strain FZB24 (006480) in May, 2000. The fact sheet noted that the strain was approved for use as a growth enhancer and disease suppressor. The invention is limited in that it teaches use of a different microorganism.
No single reference and no combination of the references teach the invention disclosed herein. The background art does not teach combinations of microorganisms disclosed herein, combinations that provide a surprising consistency of performance in plant disease control.
A purpose of the invention is to control the plant pathogens that cause early and late season root and stalk rot. Another purpose is to provide for season-long protection for plants from the pathogens that cause early and late season root and stalk rot. Another purpose is to provide consistent disease control for plants.
One advantage of the invention is that root and stalk rot can be controlled with a composition that is not toxic to humans. Another advantage of the invention is that root and stalk rot can be controlled more economically than with chemical fungicides. Yet another advantage of the invention is that it provides a biocontrol agent or bio-pesticide with extended shelf life. Thus, a seed can be treated with the biocontrol agent and stored for a period of months and still host a viable biocontrol agent that will colonize the root when the seed is placed in the ground, germinates and grows. Furthermore, the disclosed biocontrol agent is competitive with natural soil microbes that occur in the rhizosphere while providing pathogen protection for the plant. A further advantage of the invention is that the combination of a fungal/bacterial antagonist is more effective in controlling fungal pathogens in the plant rhizosphere than either a fungal antagonist or a bacterial antagonist alone, Thus, the invention provides an easy-to-use, effective means of controlling plant pathogens that have been only been controllable by rotation management. A further advantage of the invention is that its use produces more consistent results than the use of either a fungal antagonist or a bacterial antagonist alone, as shown by the Working Examples presented herein. In fact, use of the antagonist combination disclosed herein are shown to be functional when use of its individual constituent antagonists is not.
The invention is an inoculum, a seed coated with the inoculum, a plant protected with the inoculum, a method of producing the inoculum and a method of protecting a seed or a plant with the inoculum. A preferred embodiment of the inoculum comprises a combination of a fungus and a bacterium. Preferably, the fungus is a species of Trichoderma and the bacterium is a species of Bacillus, preferably a spore-forming strain of Bacillus. More preferably, the fungus is Trichoderma virens and the bacterium is Bacillus amyloliquifaciens, although other combinations are also preferred. Even more preferably, the fungus is Trichoderma virens GL-3 (ATCC 58678) and the bacterium is Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390).
In a preferred embodiment, the inoculum is produced by adding an essentially pure culture, a substantially pure culture, an axenic culture or a biologically pure culture of Trichoderma virens GL-3 to a bioreactor containing molasses-yeast extract growth medium using a standard inoculation technique. The medium is agitated and aerated and its temperature is maintained at about 28 degrees Centigrade. After the Trichoderma virens GL-3 is grown in the medium for about eight hours, an essentially pure culture, a substantially pure culture, an axenic culture or a biologically pure culture of Bacillus amyloliquefaciens TJ1000 or 1BE is added to the medium using a standard inoculation technique. The combined, competitive culture is grown under the aforementioned conditions and produces maximum cell and spore counts in approximately seven days. The combined culture is then used as an inoculum and is applied each seed at a rate of no less than about 1,000 spore counts per seed.
In another preferred embodiment, a solution containing an essentially pure culture, a substantially pure culture, an axenic culture or a biologically pure culture of the fungal antagonist Trichoderma virens GL-3 is combined with a solution containing an essentially pure culture, a substantially pure culture, an axenic culture or a biologically pure culture of Bacillus amyloliquefaciens TJ1000 or 1BE in a 50/50 mixture by volume and is applied to a seed at a rate of no less than about 10,000 spore counts per seed.
In broad terms, a preferred embodiment of the invention is an agricultural inoculum suitable for inoculating plant seeds comprising a fungal antagonist selected from the group consisting of Trichoderma virens GL-3 (ATCC 58678) and mutants thereof, a bacterial antagonist selected from the group consisting of Bacillus amyloliquefaciens TJ1000 or 1 BE (ATCC BAA-390) and mutants thereof, and a suitable carrier that is non-phytotoxic, non-bacteriostatic, and non-bacteriocidal.
Another preferred embodiment of the invention is a composition of matter comprising a plant seed inoculated with a combination comprising a fungal antagonist selected from the group consisting of Trichoderma virens GL3 (ATCC 58678) and mutants thereof and a bacterial antagonist selected from the group consisting of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and mutants thereof, wherein said combination suppresses growth of plant pathogenic fungi.
Yet another preferred embodiment of the invention is a seed or plant inoculated with a combination comprising a fungal antagonist selected from the group consisting of Trichoderma virens GL-3 (ATCC 58678) and mutants thereof and a bacterial antagonist selected from the group consisting of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and mutants thereof, wherein the combination suppresses growth of plant pathogenic fungi.
In broad terms, a preferred embodiment of the invention is a method of protecting a plant from disease caused by a plant pathogenic fungus comprising inoculating seeds from said plant with a combination comprising a fungal antagonist selected from the group consisting of Trichoderma virens GL-3 (ATCC 58678) and mutants thereof and a bacterial antagonist selected from the group consisting of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and mutants thereof, wherein said combination suppresses growth of plant pathogenic fungi.
Another preferred embodiment of the invention is a method of protecting a seed or a plant from disease caused by a plant pathogenic fungus comprising inoculating seeds from said plant with a composition comprising a spore-forming fungal antagonist and a spore-forming bacterial antagonist. Preferably, the spore-forming bacterial antagonist is selected from the group Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and mutants thereof.
Another preferred embodiment of the invention is a method of protecting a seed or a plant from disease caused by a plant pathogenic fungus comprising inoculating seeds from said plant with a composition comprising a fungal antagonist and a bacterial antagonist, wherein said combination suppresses growth of plant pathogenic fungi. A preferred embodiment is capable of control of the plant pathogen fungi Fusarium, Phythium, Phytophthora and Penicillium.
Another preferred embodiment of the invention is a method of protecting a plant from disease caused by a plant pathogenic fungus comprising inoculating seeds from said plant with a composition comprising a fungal antagonist and a bacterial antagonist selected from the group consisting of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and mutants thereof, wherein said combination suppresses growth of plant pathogenic fungi.
Yet another preferred embodiment of the invention is a method for biologically controlling or inhibiting stalk rot or root rot comprising coating seeds with an effective amount of a composition comprising Trichoderma virens GL-3 (ATCC 58678) and Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390).
A further preferred embodiment of the invention is process for making a composition comprising introducing an essentially pure culture of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) to a growth medium about eight hours after an essentially pure culture of Trichoderma virens GL-3 (ATCC 58678) is introduced to the growth medium and growing the culture as a competitive culture.
Another preferred embodiment of the invention is a process comprising making a composition by combining an essentially pure culture of Trichoderma virens GL-3 (ATCC 58678) with an essentially pure culture of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) in a 50:50 mixture and applying said composition to a seed at a rate of at least 100,000 spores per seed.
A further preferred embodiment of the invention is a method for protecting plants in a growing medium from damping off and root rot fungal plant disease comprising placing in the growing medium in the immediate vicinity of the plant to be protected an effective quantity of one of the fungal/bacterial combinations disclosed herein.
Yet another preferred embodiment of the invention is a method for protecting plants from fungal plant disease comprising adding one of the fungal/bacterial combinations disclosed herein in an effective quantity to a substrate such as pelletized calcium sulfate or pelletized lime and placing the pellet in the immediate vicinity of the plant to be protected. The pellet may or may not contain other nutrients.
Another preferred embodiment of the invention is a method for protecting plants from fungal plant disease comprising adding one of the fungal/bacterial combinations disclosed herein in an effective quantity to a liquid solution such as water and applying the liquid solution in the immediate vicinity of the plant to be protected. The liquid may or may not contain additional nutrients and may include a chemical fungicide applied to the seed such as, for example, Maxim or captan. The disclosed combination may also be added to a plant nutrient (nitrogen-phosphorus-potassium (NPK)) plus plant micro-nutrient solution that is compatible with the combination and applied as an in-furrow treatment.
A further preferred embodiment of the invention is a method for biologically controlling a plant disease caused by a plant-colonizing fungus, the method comprising inoculating a seed of the plant with an effective amount of a microbial inoculant comprising a combination of microorganisms having all of the identifying characteristics of Trichoderma virens Gl-3 and Bacillus amyloliquefaciens TJ1000 or 1BE, said inoculation resulting in the control of said plant disease. The invention is also a method according to the above preferred embodiment wherein said inoculation results in the control of more than one plant disease.
Yet another preferred embodiment of the invention involves combining of a spore forming fungal strain and a spore forming bacterial strain to enhance ease of use and longevity of shelf life both as a stored product and when applied to a seed. In another preferred embodiment, the invention involves applying the disclosed Trichoderma microorganism and the Bacillus microorganism to a wettable powder, in which form it is applied.
Another preferred embodiment of the invention is composition of matter made by combining: a fungal antagonist selected from the group consisting of Trichoderma virens GL-3 (ATCC 58678) and mutants thereof; a bacterial antagonist selected from the group consisting of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and mutants thereof; and a suitable carrier that is non-phylotoxic, non-bacteriostatic, and non-bacteriocidal.
A further preferred embodiment of the invention is an antagonist for controlling plant pathogens made by combining effective amounts of: a fungal antagonist selected from the group consisting of Trichoderma virens GL-3 (ATCC 58678) and mutants thereof; a bacterial antagonist selected from the group consisting of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and mutants thereof; and a suitable carrier that is non-phytotoxic, non-bacteriostatic, and non-bacteriocidal.
Yet another preferred embodiment of the invention is a seed assembly made by combining a plant seed with effective amounts of a spore-forming bacterial antagonist and a spore-forming fungal antagonist. In a preferred embodiment, the seed is a seed of a plant selected from the group consisting of a monocot, and a dicot. In another preferred embodiment, the seed is a seed of a plant selected from the group consisting of a legume plant, and a non-legume plant. In another preferred embodiment, the seed is a seed of a plant selected from the group consisting of corn, sunflower, soybean, field pea, and wheat.
Another preferred embodiment of the invention is method for culturing a plant comprising: applying an antagonist disclosed herein to a seed or to the seedbed of the plant; planting the seed in the seedbed; growing the plant to yield a crop; and harvesting the crop; wherein said applying step increases the yield of the crop. In another preferred embodiment, the antagonist is applied to the seed or to the seedbed of a plant selected from the group consisting of a monocot, and a dicot. In another preferred embodiment, the antagonist is applied to the seed or to the seedbed of a plant selected from the group consisting of a legume plant, and a non-legume plant. In another preferred embodiment, the antagonist is applied to the seed or to the seedbed of a plant selected from the group consisting of corn, sunflower, soybean, field pea, and wheat.
Yet another preferred embodiment is a process comprising: making a composition by combining an essentially pure culture of Trichoderma virens GL-3 (ATCC 58678) with an essentially pure culture of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) in a mixture; and applying said composition to a seed; wherein said mixture ranges in composition from 10 to 90 percent Trichoderma virens GL-3 (ATCC 58678) by volume and from 90 to 10 percent Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) by volume.
Yet another preferred embodiment of the invention is a process comprising: making a composition by combining an essentially pure culture of Trichoderma virens GL-3 (ATCC 58678) with a plurality of essentially pure cultures of bacteria in a mixture; and applying said composition to a seed; wherein said mixture ranges in composition from 10 to 90 percent Trichoderma virens GL-3 (ATCC 58678) by volume.
A further preferred embodiment of the invention is an antagonist for controlling plant pathogens made by combining effective amounts of: a fungal antagonist selected from the group consisting of a strain of Trichoderma virens and mutants thereof; a bacterial antagonist selected from the group consisting of Bacillus amyloliquefaciens TJ1000 or 1BE (ATCC BAA-390) and mutants thereof; and a suitable carrier that is non-phytotoxic, non-bacteriostatic, and non-bacteriocidal. Preferably, the strain is Trichoderma viren Gl 21, which is presently EPA registered.
In a preferred embodiment, the invention is an antagonist for controlling plant pathogens made by combining effective amounts of: a fungal antagonist selected from the group consisting of Trichoderma virens GL-3 (ATCC 58678) and mutants thereof; a plurality of bacterial antagonists; and a suitable carrier that is non-phytotoxic, non-bacteriostatic, and non-bacteriocidal. Preferably, the plurality of bacterial antagonists comprises a strain of Erwinia carotovora and/or a strain of Bacillus lentimorbus. 
Further aspects of the invention will become apparent from consideration of the drawings and the ensuing description of preferred embodiments of the invention. A person skilled in the art will realize that other embodiments of the invention are possible and that the details of the invention can be modified in a number of respects, all without departing from the inventive concept. Thus, the following drawings and description are to be regarded as illustrative in nature and not restrictive.